Molecular communication: An acid tale of prion formation

Some bacteria use lactic acid to communicate with yeast cells

Study of immunological responses of mice immunized with DNA vaccines targeting specific tumor associated antigens

Cancer is a leading cause of mortality worldwide, and conventional therapies, including surgery, radiation and chemotherapy are highly invasive without offering lifelong protection. Nowadays it is believed that the immune system is one of the major players in cancer development, since it became clear that tumors evade immune surveillance and destruction and they co-opt certain immune-checkpoint pathways as a major mechanism of immune resistance, particularly against T cells specific for tumor antigens. Thus, immunotherapy has received increasing attention over the last few decades as a strategy for cancer treatment and new immunotherapeutic approaches have been developed that are positively influencing clinical outcome for cancer patients. The goal of immunotherapy is to harness the immune system to elicit specific immune responses against tumor-associated antigens (TAAs). In this context, DNA vaccination represents a promising strategy for harnessing the immune system to fight cancer. DNA vaccines are easy to construct and store, low-priced, safe and reproducible. Cancer DNA vaccines are designed to deliver one or several genes encoding tumor antigens or to modulate immune responses, thereby eliciting or augmenting immune responses against tumor antigens that play central role in tumor initiation, progression and metastasis. Vaccine efficacy can be significantly improved not only through selection of appropriate tumor antigens, moreover, by implementing strategies for improving antigen presentation and immunogenicity. Cancer DNA vaccines can induce both innate immunity activation and adaptive immune responses in order to suppress tumor growth and achieve total tumor rejection. New developments in vaccine delivery methods improve the efficiency of tumor antigens to evoke immune responses with the aim to overcome the primary hurdle of poor transfection efficacy and subsequent lower immunogenicity. DNA vaccines are usually administered intramuscularly or intradermal. Muscle and skin cells are able to accumulate antigens, but unable to prime immune responses. Recombinant plasmids that enter somatic cells are directed to the nucleus and express the encoded genes. Produced antigens are presented through MHC class I molecules to CD8 T cells. In intramuscular administration, most of the vaccine is received by the muscle cells. Synthesized proteins are released in the extracellular matrix and are endocytosed or phagocytosed and displayed by antigen-presenting cells through MHC class II molecules to activate CD4 T helper cells in the presence of costimulatory signals. Activated B cells differentiate into plasma cells and secrete antibodies that bind antigenic epitopes. The antigenic peptides that have remained in the cytoplasm of the targeted cells are initially presented via MHCI molecules and activate cytotoxic CD8 T cells. When the vaccine is administered intradermally, DNA enters the skin and dendritic cells, such as Langerhans cells, present antigens. Death of the antigen expressing cells increases antigen presentation and causes enhanced Th1 response.The present study aimed to design, develop and evaluate a new DNA vaccine to treat breast adenocarcinoma in a syngeneic experimental cancer model in BALB/c mice. Among the variety of tumor associated antigens, BIRC5, EpCAM and the novel NPTN were initially selected. DNA vaccine vectors were constructed and the selected antigens were expressed in cancer cell lines in vitro and in murine tissue in vivo. Our first experimental data and extensive literature search resulted in the selection of the cell adhesion molecule NPTN -neuroplastin- as a new promising tumor antigen.Neuroplastin is a transmembrane glycoprotein which exists in two isoforms. It belongs to the immunoglobulin superfamily of cell adhesion molecules (CAMs). In adults, NPTN is expressed almost exclusively in the immune "privileged" site of brain. Neuroplastin was recently identified as a novel breast cancer tumor associated antigen. Additionally, neuroplastin expression was detected in breast cancer cell lines and breast biopsies of advanced stage carcinomas.In order to examine the protective effect of neuroplastin DNA vaccine against breast adenocarcinoma, an experimental BALB/c syngeneic breast cancer model was established. 5x106 DA3 murine breast cancer cells were injected subcutaneously and tumor development was observed. One week later mice were euthanized; blood, tumor and spleen were collected. Tumor volume was calculated by the use of the modified ellipsoid formula (length*width2)/2. Neuroplastin protective efficacy was evaluated upon intra ear pinna vaccination. Vaccination (50mg) was performed for three times with 7 days interval. Mice were challenged with DA3 cells 3-5 days following the last immunization.DNA vaccines encoding neuroplastin, elicited protective immunity in BALB/c mice. Most NPTN-immunized mice did not develop tumors or if so, the developed tumors had statistically lower tumor volume compared to non-vaccinated mice or mice vaccinated with the vector alone. Interestingly, tumors isolated from immunized mice presented low expression level of the tumor antigens EpCAM and NPTN compared to control mice.The Neuroplastin DNA vaccine successfully raised antigen-specific immune responses in immunized BALB/c mice. NPTN-specific antibodies were detected both by confocal fluorescence microscopy and ELISA following immunization of mice (before or after tumor challenge). Sera were assayed as a source of primary anti-NPTN antibodies. Specific antibody titer in sera from immunized mice was comparable to the titer of the commercially available anti-NPTN antibody.Furthermore, the neuroplastin-specific immune responses were evaluated. Spleens were harvested from immunized mice and splenocytes were cultured and stimulated with four different antigenic neuroplastin peptides or their combination. Peptides were selected based on their capability to be presented by MHCI molecules to CD8+ T cells. Therefore 9mer peptides were selected using SYFPEITHI and IEDB databases designed for presentation by H2-Kd and H2-Ld MHC molecules of BALB/c mice. Culture supernatant of stimulated splenocytes was assayed by ELISPOT and IFN-γ production from many different clones of antigen-specific CD8+ cells was detected. The experimental data support the development of a CD8+ T cell cytotoxic immune response. Analysis of the antibody isotypes of sera from immunized or non-immunized BALB/c mice showed an increase in IgG2b and IgG3 isotype levels and a reduction in IgG1 antibodies. The observed immunoglobulin class switch is associated with a Th1 immune response. In addition, IL-12/IL-23 p40 and TNFa levels were significantly increased in the group of NPTN immunized mice, as confirmed by ELISA. Flow cytometry experiments revealed high CD8+ T cell infiltration of tumors and spleens of the immunized mice, which is consistent with IFN-γ production, cytotoxic response and the overall cytokine expression pattern.Our data suggest that a neuroplastin encoding DNA vaccine offer protective immunity in BALB/c mice with syngeneic breast adenocarcinoma without signs of autoimmunity. Mice raised NPTN-specific antibodies and vaccination promoted a cytotoxic Th1 immune response, as documented by isotype switch, cytokine profile and tumor infiltration of lymphocytes.Further investigation of neuroplastin role and the molecular signaling pathway could lead to the discovery of important target molecules involved in carcinogenesis. Early experiments showed that in vitro overexpression of neuroplastin leads to transcriptional activation of genes associated with angiogenesis and metastasis. Also, using an in vitro migration assay we showed that NPTN overexpression increased the migratory potential of HeLa cancer cells. The ultimate goal of our study is to further translate our findings into clinical practice in order to develop effective and specific protective immunity against mammary carcinoma in breast cancer patients.Τα DNA εμβόλια αποτελούν σημαντικό εργαλείο για την καταπολέμηση του καρκίνου με ανοσοθεραπευτικές προσεγγίσεις παρέχοντας μεγάλη ευελιξία και ασφάλεια. Η παρούσα μελέτη είχε ως στόχο τον σχεδιασμό, την ανάπτυξη και αξιολόγηση ενός νέου DNA γενετικού εμβολίου με σκοπό την αντιμετώπιση του αδενοκαρκινώματος μαστού σε συνγονικό πειραματικό μοντέλο καρκίνου σε BALB/c ποντικούς. Ανάμεσα στην πληθώρα των ογκοσχετιζόμενων καρκινικών αντιγόνων, αρχικά επιλέχθηκαν τα αντιγόνα BIRC5, EpCAM και NPTN. Στην παρούσα μελέτη επιτεύχθηκε η δημιουργία DNA εμβολίων ικανών να εκφράζονται σε κυτταρικές σειρές in vitro και σε ιστούς in vivo. Τα αρχικά πειραματικά δεδομένα και η εκτεταμένη βιβλιογραφική αναζήτηση οδήγησαν στο προσκολλητικό μόριο NPTN –νευροπλαστίνη- ως ένα νέο πολλά υποσχόμενο αντιγόνο των όγκων. Η νευροπλαστίνη είναι μία διαμεμβρανική γλυκοπρωτεΐνη, που απαντάται σε δύο ισομορφές. Ανήκει στην υπεροικογένεια των ανοσοσφαιρινών των προσκολλητικών μορίων CAMs. Σε ενήλικα άτομα, η νευροπλαστίνη εκφράζεται σχεδόν αποκλειστικά στην ανοσολογικά «προνομιούχα» περιοχή του εγκεφάλου. Η νευροπλαστίνη ταυτοποιήθηκε πρόσφατα ως καρκινικό αντιγόνο του καρκίνου του μαστού. Μάλιστα η έκφρασή της εντοπίστηκε σε καρκινικές κυτταρικές σειρές μαστού και βιοψίες καρκινωμάτων μαστού προχωρημένου σταδίου. Κατά τη διάρκεια της μελέτης του DNA εμβολίου της νευροπλαστίνης ανιχνεύθηκε αντιγονο-ειδική ανοσολογική απόκριση σε BALB/c ποντικούς. Τα NPTN-ειδικά αντισώματα ανιχνεύθηκαν με συνεστιακή μικροσκοπία φθορισμού και ELISA μετά από ανοσοποίηση των ποντικών τόσο πριν, όσο και μετά την πρόκληση συνγονικού αδενοκαρκινώματος του μαστού. Ο εμβολιασμός (50μg) πραγματοποιήθηκε 3 φορές ανά διάστημα 7 περίπου ημερών intra ear. Η πλειονότητα των NPTN-ανοσοποιημένων ποντικών δεν εμφάνισαν όγκο ή το μέγεθος του όγκου ήταν στατιστικά μικρότερο από αυτό των ποντικών που δεν εμβολιάστηκαν ή εμβολιάστηκαν με τον φορέα κλωνοποίησης μόνο. Τα δεδομένα της έρευνάς μας υποστηρίζουν την ανάπτυξη κυτταροτοξικής ανοσολογικής απόκρισης. Τα σπληνοκύτταρα από ανοσοποιημένους ποντικούς καλλιεργήθηκαν και διεγέρθηκαν με 4 διαφορετικά αντιγονικά πεπτίδια και το συνδυασμό τους. Τα πεπτίδια αυτά επιλέχθηκαν με σκοπό την βέλτιστη παρουσίασή τους από MHCI μόρια στα CD8+ κύτταρα. Γι΄ αυτό επιλέχθηκαν 9μερή πεπτίδια (παρουσίαση στον TCR των κυτταροτοξικών κυττάρων) μετά από αναζήτηση στις βάσεις δεδομένων SYFPEITHI και IEDB για τα MHC μόρια των BALB/c ποντικών H2-Kd και H2-Ld. Το υπερκείμενο της καλλιέργειας των διεγερμένων σπληνοκυττάρων υποβλήθηκε σε δοκιμή ELISPOT και έδειξε παραγωγή υψηλών επιπέδων IFN-γ από πολλούς διαφορετικούς CD8 κλώνους κυττάρων. Η ανάλυση των ισοτύπων των αντισωμάτων στον ορό ανοσοποιημένων και μη BALB/c ποντικών έδειξε αύξηση των IgG2b και IgG3 αντισωμάτων και μείωση των IgG1b αντισωμάτων. Η μεταστροφή αυτή των ισοτύπων σχετίζεται με Th1 ανοσολογική απόκριση. Επιπροσθέτως, τα επίπεδα της IL-12 /IL-23 p40 και του TNFa σχεδόν διπλασιάστηκαν στους ανοσοποιημένους ποντικούς, όπως επιβεβαιώθηκε με ELISA. Πειράματα κυτταρομετρίας ροής αποκάλυψαν υψηλή συγκέντρωση CD8+ T λεμφοκυττάρων στους όγκους και τους σπλήνες των ανοσοποιημένων ποντικών, γεγονός που συνάδει με την παραγωγή IFN-γ και το συνολικό πρότυπο έκφρασης των κυτταροκινών. Η περαιτέρω διερεύνηση της δράσης της νευροπλαστίνης και του μοριακού μονοπατιού σηματοδότησής της μπορεί να οδηγήσει στην ανακάλυψη σημαντικών μορίων στόχων που ενέχονται στην καρκινογένεση. Πρώιμα πειράματα έδειξαν ότι η in vitro υπερέκφραση της νευροπλαστίνης οδηγεί στην μεταγραφική ενεργοποίηση γονιδίων που σχετίζονται με την αγγειογένεση και την μετάσταση. Επίσης in vitro δοκιμή μετάστασης έδειξε ότι η υπερέκφραση της νευροπλαστίνης στην καρκινική κυτταρική σειρά HeLa αυξάνει το μεταναστευτικό δυναμικό των κυττάρων. Απώτερος στόχος είναι η μετάφραση των ευρημάτων μας στην κλινική πράξη με σκοπό την ανάπτυξη αποτελεσματικής προστατευτικής ανοσίας σε ασθενείς με καρκίνο του μαστού

Dietary mastic oil extracted from Pistacia lentiscus var. chia suppresses tumor growth in experimental colon cancer models

Plant-derived bioactive compounds attract considerable interest as potential chemopreventive anticancer agents. We analyzed the volatile dietary phytochemicals (terpenes) present in mastic oil extracted from the resin of Pistacia lentiscus var. chia and comparatively investigated their effects on colon carcinoma proliferation, a) in vitro against colon cancer cell lines and b) in vivo on tumor growth in mice following oral administration. Mastic oil inhibited - more effectively than its major constituentsproliferation of colon cancer cells in vitro, attenuated migration and downregulated transcriptional expression of survivin (BIRC5a). When administered orally, mastic oil inhibited the growth of colon carcinoma tumors in mice. A reduced expression of Ki-67 and survivin in tumor tissues accompanied the observed effects. Notably, only mastic oil -which is comprised of 67.7% α-pinene and 18.8% myrceneinduced a statistically significant anti-tumor effect in mice but not α-pinene, myrcene or a combination thereof. Thus, mastic oil, as a combination of terpenes, exerts growth inhibitory effects against colon carcinoma, suggesting a nutraceutical potential in the fight against colon cancer. To our knowledge, this is the first report showing that orally administered mastic oil induces tumor-suppressing effects against experimental colon cancer

Evaluation of Antioxidant and Antiproliferative Properties of Cornus mas L. Fruit Juice

Cornus mas L. (Cornelian cherry) is a flowering plant indigenous to Europe and parts of Asia, mostly studied for the antimicrobial activity of its juice. In this report, we investigated the composition and the in vitro antioxidant capacity of Cornus mas L. fruit juice from Greece, as well as its antiproliferative properties in vitro and in vivo. The fruits showed a high content of citric, malic, and succinic acid, in contrast to their juice, which had a low concentration of organic acids. The juice demonstrated significant antioxidant activity against the free radical 2,2-diphenyl-1-picrylhydrazyl (DPPH) and modest antiproliferative potential against four human cancer cells lines and one murine: mammary adenocarcinoma MCF-7, hepatocellular carcinoma HepG2 and colon adenocarcinomas Caco2, HT-29, as well as murine colon carcinoma CT26. Cell viability was reduced by 40–50% following incubation of the cells with the highest concentration of the juice. Although Cornelian cherry juice exhibited in vitro growth inhibitory effects against colon carcinoma cells, no tumor growth inhibition was observed in an in vivo experimental colon carcinoma model in mice following prophylactic oral administration of a daily dose of 100 μL juice for a period of 10 days. Thus, our findings raise interesting questions for further research on Cornus mas L. fruit juice, and in parallel, the strong antioxidant potential implies that the plant could be further explored and exploited for its protective effect against oxidative damage

Phytochemical profile and evaluation of the biological activities of essential oils derived from the greek aromatic plant species Ocimum basilicum, Mentha spicata, Pimpinella anisum and Fortunella margarita

Natural products, known for their medicinal properties since antiquity, are continuously being studied for their biological properties. In the present study, we analyzed the composition of the volatile preparations of essential oils of the Greek plants Ocimum basilicum (sweet basil), Mentha spicata (spearmint), Pimpinella anisum (anise) and Fortunella margarita (kumquat). GC/MS analyses revealed that the major components in the essential oil fractions, were carvone (85.4%) in spearmint, methyl chavicol (74.9%) in sweet basil, trans-anethole (88.1%) in anise, and limonene (93.8%) in kumquat. We further explored their biological potential by studying their antimicrobial, antioxidant and antiproliferative activities. Only the essential oils from spearmint and sweet basil demonstrated cytotoxicity against common foodborne bacteria, while all preparations were active against the fungi Saccharomyces cerevisiae and Aspergillus niger. Antioxidant evaluation by DPPH and ABTS radical scavenging activity assays revealed a variable degree of antioxidant potency. Finally, their antiproliferative potential was tested against a panel of human cancer cell lines and evaluated by using the sulforhodamine B (SRB) assay. All essential oil preparations exhibited a variable degree of antiproliferative activity, depending on the cancer model used, with the most potent one being sweet basil against an in vitro model of human colon carcinoma

Evaluating the value of day 0 of an ICSI cycle on indicating laboratory outcome

A number of oocyte characteristics have been associated with fertilization, implantation and live-birth rates, albeit without reaching a consensus. This study aims to delineate possible associations between oocyte characteristics, oocyte behavior during intracytoplasmic sperm injection (ICSI), fertilization potential, and laboratory outcomes. Four-hundred and seventy-seven patients, yielding 3452 oocytes, were enrolled in this prospective observational study from 2015 to 2018. Οoplasm granularity was associated with poor embryo quality and higher probabilities of post-ICSI oocytes and embryos discarded in any developmental stage and never selected for embryo transfer or cryopreservation (p < 0.001). Both sudden or difficult ooplasm aspiration, and high or lack of resistance during ICSI were associated with either a poor Zygote-Score or fertilization failure (p < 0.001). Sudden or difficult ooplasm aspiration and high resistance during ICSI penetration were positively associated with resulting to a post-ICSI oocyte or embryo that would be selected for discard. Evaluation of oocyte characteristics and oocyte behavior during ICSI may provide early information regarding laboratory and cycle outcomes. Particularly, ooplasm granularity, and fragmentation of polar body, along with sudden or difficult ooplasm aspiration and high or lack of resistance during ICSI penetration may hinder the outcome of an ICSI cycle. The associations presented herein may contribute towards development of a grading system or a prediction model. Taking into account information on oocytes and ICSI behavior may effectively assist in enhancing IVF outcome rates. © 2020, The Author(s)

Lactobacillus casei Exerts Anti-Proliferative Effects Accompanied by Apoptotic Cell Death and Up-Regulation of TRAIL in Colon Carcinoma Cells.

Probiotic microorganisms such as lactic acid bacteria (LAB) exert a number of strain-specific health-promoting activities attributed to their immunomodulatory, anti-inflammatory and anti-carcinogenic properties. Despite recent attention, our understanding of the biological processes involved in the beneficial effects of LAB strains is still limited. To this end, the present study investigated the growth-inhibitory effects of Lactobacillus casei ATCC 393 against experimental colon cancer. Administration of live Lactobacillus casei (as well as bacterial components thereof) on murine (CT26) and human (HT29) colon carcinoma cell lines raised a significant concentration- and time-dependent anti-proliferative effect, determined by cell viability assays. Specifically, a dramatic decrease in viability of colon cancer cells co-incubated with 10(9) CFU/mL L. casei for 24 hours was detected (78% for HT29 and 52% for CT26 cells). In addition, live L. casei induced apoptotic cell death in both cell lines as revealed by annexin V and propidium iodide staining. The significance of the in vitro anti-proliferative effects was further confirmed in an experimental tumor model. Oral daily administration of 10(9) CFU live L. casei for 13 days significantly inhibited in vivo growth of colon carcinoma cells, resulting in approximately 80% reduction in tumor volume of treated mice. Tumor growth inhibition was accompanied by L. casei-driven up-regulation of the TNF-related apoptosis-inducing ligand TRAIL and down-regulation of Survivin. Taken together, these findings provide evidence for beneficial tumor-inhibitory, anti-proliferative and pro-apoptotic effects driven by this probiotic LAB strain

Oral administration of live <i>Lactobacillus casei</i> inhibits <i>in vivo</i> growth of colon carcinoma tumors in mice.

<p>(A) Schematic representation of the <i>in vivo</i> tumor model. Live <i>L</i>. <i>casei</i> was administered <i>per os</i> daily to BALB/c mice for 13 days. At day 10, 5x10⁶ CT26 cells were inoculated subcutaneously. Mice in the control group received PBS. Tumors were harvested from euthanized animals 7 days after administration of CT26 cells. (B) Mean tumor volume of tumors excised from mice that received <i>L</i>. <i>casei</i> (LC) or PBS (control). A statistically significant (<i>p</i> < 0.001) reduction of ≈80% in tumor volume was observed in <i>L</i>. <i>casei</i>-treated mice as compared to control. (C) Photographic observation of tumors harvested from control (PBS)- or <i>L</i>. <i>casei</i> (LC)-treated mice. (D) Immunohistochemical detection of TRAIL (Di, Diii) and Survivin (Dii, Div) in CT26 tumors in BALB/c mice. Detection of TRAIL in tumor tissue from <i>L</i>. <i>casei</i>-treated (Diii) compared to control (Di) mice, and Survivin in tumor tissue from <i>L</i>. <i>casei</i>-treated (Div) or control (Dii) mice. At least ten sections per tumor and seven tumors per group from two independent experiments were analyzed. The difference in the expression of TRAIL in tumor tissue from treated (Diii) versus control (Di) mice is statistically significant (<i>p</i> = 0.007), as calculated using the SigmaPlot 11.0 statistical software.</p